The process of how proteins are segregated and sorted in the trans-Golgi Network and endosomal system of eukaryotic cells is one of the fundamental mechanisms by which cells can establish and maintain many complex specialized functions such as polarization, phagocytosis, and regulated secretion. Identifying the protein machinery that controls these pathways as well as functionally determining how this machinery operates and how it might be regulated will be the key to understanding this general process. One model system for addressing these issues has been the study of how proteins traffic to the lysosome (vacuole) of Saccharomyces cerevisiae. Previous studies have uncovered a new protein trafficking pathway that transports a subset of vacuolar proteins from the trans-Golgi Network. This pathway bypasses transit through the endosomal/prevacuolar compartment. Protein such as Alkaline Phosphatase and the vacuolar syntaxin Vam3p do not enter into secretory vesicles bound for the plasma membrane or TGN-derived vesicles bound for the endosomal prevacuolar compartment. Instead, these proteins appear to enter a new class of vesicles that may fuse directly with the vacuole. This sorting event is mediated by determinants within the cytosolic tail of these proteins. So far, the dynamin-like protein Vps1p and the non-clathrin associated adaptor complex AP3 have been found to participate in this process. Importantly, both Vps1p and AP3 have close homologues in mammalian cells indicating that this novel trafficking pathway operates in all eukaryotic cells. The goal of this proposal will be to identify and functionally characterize the proteins that control the selective transport of Alkaline Phosphatase to the vacuole along this new vacuolar biogenesis pathway using an approach that combines genetics and biochemistry. Genetic screens will be conducted to identify the components required for the specific sorting of ALP in the TGN. A functional analysis of the corresponding proteins will then be conducted to delineate the protein:protein interactions that selectively package ALP into its cognate transport intermediate. These studies will also identify and functionally characterize the genes required for the fusion of ALP- containing vesicles with the vacuole. Finally, conditional alleles of the genes required for the fusion of ALP-containing vesicles with the vacuole will be exploited to isolate these membrane trafficking intermediates and analyze their constituents.